Clinical Trials

Day of Embryo Transfer for Patients Undergoing In Vitro FertilizationNot Recruiting

We are examining whether pregnancy rates differ based on day of embryo transfer in patients
who replace all available embryos after an IVF cycle. Patients must be undergoing IVF
treatment at Stanford University and patients will not receive compensation for their
participation (no medical costs covered or patient payment for participation).

Stanford is currently not accepting patients for this trial.For more information, please contact Lora Shahine, (650) 498 - 7911.

Abstract

Previous studies have demonstrated that aneuploidy in human embryos is surprisingly frequent with 50-80% of cleavage-stage human embryos carrying an abnormal chromosome number. Here we combine non-invasive time-lapse imaging with karyotypic reconstruction of all blastomeres in four-cell human embryos to address the hypothesis that blastomere behaviour may reflect ploidy during the first two cleavage divisions. We demonstrate that precise cell cycle parameter timing is observed in all euploid embryos to the four-cell stage, whereas only 30% of aneuploid embryos exhibit parameter values within normal timing windows. Further, we observe that the generation of human embryonic aneuploidy is complex with contribution from chromosome-containing fragments/micronuclei that frequently emerge and may persist or become reabsorbed during interphase. These findings suggest that cell cycle and fragmentation parameters of individual blastomeres are diagnostic of ploidy, amenable to automated tracking algorithms, and likely of clinical relevance in reducing transfer of embryos prone to miscarriage.

Abstract

Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility treatment and future derivation of patient-specific embryonic stem cells.

Abstract

Preimplantation genetic diagnosis (PGD) is an increasingly common adjunct to IVF. The information gained from PGD may be used to reduce the incidence of chromosomally abnormal pregnancies and augment the current selection process of embryos. As such, patients may choose to utilize PGD in either fresh or cryopreserved IVF cycles. It is a common practice to cryopreserve excess embryos at the blastocyst stage. In these cases, trophectoderm biopsy is the only technique available for PGD. This articles reports this study centre's experience with trophectoderm biopsies of cryopreserved blastocysts in 12 patients who underwent 13 cycles of PGD. The implantation rate per embryo transferred was 46% and the ongoing pregnancy rate per embryo transfer was 63%. The results from this case series demonstrate that trophectoderm biopsy on cryopreserved blastocysts to perform PGD is logistically feasible. In addition, the rate of implantation and ongoing pregnancy were maintained within a reasonable range to justify the procedure. Preimplantation genetic diagnosis (PGD) is an increasingly common adjunct to IVF and is used to evaluate the genetic makeup of the embryo prior to transfer of the embryo into the uterus. The information gained from PGD may be used to identify single-gene disorders that result in genetic disease, reduce the incidence of chromosomally abnormal pregnancies and/or augment the selection process of embryos to be transferred. In order to perform PGD, a biopsy of the embryo is the performed and cells are removed for testing. PGD may be performed in either fresh or frozen (cryopreserved) IVF cycles. Patients who have cryopreserved embryos remaining in storage from a previous fresh cycle may wish to have these embryos tested with PGD. Many embryos are frozen on day 5 of development, referred to as the blastocyst stage. At this stage of development, embryo biopsy is performed via a technique known as 'trophectoderm biopsy', in which 1-3 of the cells destined to become the placenta are removed from the embryo for chromosomal testing. We report our experience with trophectoderm biopsy of frozen blastocysts in 12 patients who underwent 13 cycles of PGD. The implantation rate per embryo transferred was 46% and the ongoing pregnancy rate per embryo transfer was 63%. The results from this case series demonstrate that trophectoderm biopsy on cryopreserved blastocysts to perform PGD is logistically feasible. In addition, the rate of implantation and ongoing pregnancy were maintained within a reasonable range to justify the procedure.

Abstract

Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.

Abstract

Over the past 20 years, numerous techniques have enhanced assisted reproductive technology outcomes to help couples have >60,000 infants in the United States in 2008. Several different days for embryo transfers have been studied, but debate for the best timing of embryo transfer is still ongoing. With growing concern about multiple gestations and neonatal outcomes, early cleavage stage embryo transfer with novel embryo selection tools may be attractive to some patients and in vitro fertilization programs. In this review, we summarize clinical and basic studies relating to the timing of embryo transfer and highlight the possibilities of safe embryo transfer by combining advanced embryo screening tools with potentially high efficiency and low adverse effects on clinical outcome.

Abstract

This prospective cohort study of infertility patients compared testosterone concentrations in early pregnancy in infertility patients who conceived naturally or after treatment. Although all groups demonstrated some increase in pregnancy testosterone from baseline concentrations, subjects who conceived following ovulation induction showed a significantly increased rise in testosterone as compared with controls (P<0.01).

Abstract

Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an extracellular matrix protein. MFS is inherited as an autosomal dominant trait and displays major manifestations in the ocular, skeletal, and cardiovascular systems. Here we report molecular and phenotypic profiles of skeletogenesis in tissues differentiated from human embryonic stem cells and induced pluripotent stem cells that carry a heritable mutation in FBN1. We demonstrate that, as a biological consequence of the activation of TGF-? signaling, osteogenic differentiation of embryonic stem cells with a FBN1 mutation is inhibited; osteogenesis is rescued by inhibition of TGF-? signaling. In contrast, chondrogenesis is not perturbated and occurs in a TGF-? cell-autonomous fashion. Importantly, skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells derived independently from MFS patient fibroblasts. Results indicate a unique phenotype uncovered by examination of mutant pluripotent stem cells and further demonstrate the faithful alignment of phenotypes in differentiated cells obtained from both human embryonic stem cells and induced pluripotent-stem cells, providing complementary and powerful tools to gain further insights into human molecular pathogenesis, especially of MFS.

Abstract

Despite the fact that the fundamental principle underlying the most common method of culture media constitution is that of mimicking the natural environment of the preimplantation embryo, one major difference that remains between current embryo culture media and in vivo conditions is the absence of growth factors in vitro. Numerous growth factors are known to be present in the in vivo environment of human and nonhuman preimplantation embryos, often with peak concentrations corresponding to when fertilization and preimplantation embryo growth would occur. Although these growth factors are found in very small concentrations, they have a profound effect on tissue growth and differentiation through attachment to factor-specific receptors on cell surfaces. Receptors for many different growth factors have also been detected in human preimplantation embryos. Preimplantation embryos themselves express many growth factors. The growth factors and receptors are metabolically costly to produce, and thus their presence in the environment of the preimplantation embryo and in the embryo respectively strongly implies that embryos are designed to encounter and respond to the corresponding factors. Studies of embryo coculture also indirectly suggest that growth factors can improve in vitro development. Several animal and human studies attest to a probable beneficial effect of addition of growth factors to culture media. However, there is still ambiguity regarding the exact role of growth factors in embryonic development, the optimal dose of growth factors to be added to culture media, the combinatorial effect and endocrine of growth factors in embryonic development.

Abstract

Using donated human embryos for scientific research raises ethical questions about the donation process. We describe a two-stage consent process designed to help couples make informed decisions about embryo disposition. This consent methodology minimizes conflict of interest, respects patient choice, and provides a much-needed resource to patients and the research community.

Abstract

Day 2 embryo transfer has been suggested as a method to improve pregnancy rates in poor responders compared with day 3 transfer. Our prospective randomized controlled trial does not show a difference in outcomes based on day of embryo transfer.

Abstract

We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.

Abstract

Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis.Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1.Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer.

Abstract

To report a case of successful pregnancy after trophectoderm biopsy and fluorescence in situ hybridization (FISH) revealed a tetraploid karyotype.Case report.A university medical center.An infertility patient desiring trophectoderm biopsy on frozen blastocysts to facilitate preimplantation genetic screening.Frozen blastocysts were thawed on the evening before transfer. Trophectoderm biopsy was performed the following morning. FISH results were available the same day, and two embryos with tetraploid results were transferred.Chorionic villus sample (CVS) and newborn exam.Normal diploid CVS result and a healthy male infant.Although multiple cells can be analyzed using trophectoderm biopsy, abnormalities in the trophectoderm may not be present in the inner cell mass.

Abstract

This review will address the current understanding of the relationship between prolactin (PRL) and endometriosis-associated infertility. Although the exact mechanisms of action of hyperprolactinemia in patients with endometriosis-associated infertility have not been clearly established, this report reviews results from relevant studies in the literature. These include serum PRL levels in endometriosis-associated infertility, PRL receptors in ectopic endometriotic tissues, basal PRL levels after TSH and Danazol (isoxazolic derivative of the synthetic steroid 5alpha-ethinyl-testosterone) therapy, peritoneal fluid and nocturnal serum PRL levels in endometriosis, infertility, and luteal phase PRL concentrations in patients with endometriosis.Obstetricians & Gynecologists, Family Physicians.After completion of this article, the reader should be able to explain the relationship between prolactin- and endometriosis-associated infertility, relate endometriosis with infertility, and summarize two ways in which prolactin and endometriosis may be linked in the pathophysiology of infertility.

Abstract

To report a case of a successful pregnancy after trophectoderm biopsy and three-probe fluorescent in situ hybridization of a frozen blastocyst.Techniques and instrumentation.A University Medical Center.Infertility patient desiring trophectoderm biopsy on frozen blastocyst for preimplantation testing, from an IVF cycle at a referring IVF program.Frozen blastocysts were thawed the evening before the planned transfer. Trophectoderm biopsy was performed in the morning. The fluorescent in situ hybridization results were obtained the same day; embryo transfer was performed under ultrasound guidance.Serum betahCG and transvaginal ultrasound.Positive betahCG and ongoing pregnancy.Trophectoderm biopsy can be used as a means for testing frozen blastocysts in patients with excess embryos cryopreserved on day 5 or 6 from previously preformed IVF cycles.

Abstract

To investigate the hypothesis that surgical treatment of endometriosis in infertile patients may improve pregnancy rates by improving embryo quality.We conducted a retrospective evaluation of 30 infertile patients treated with in vitro fertilization (IVF) before and after surgery for endometriosis. Patients served as their own controls and only cycles with similar stimulation protocols were compared.Using standard visual evaluation, embryo quality on day 3 was similar before and after surgical treatment of endometriosis. Fifty seven percent of patients had stage I-II endometriosis and 43% had stage III-IV disease. No patients had a live birth after the first IVF cycle and 43% of patients had a live birth with the IVF cycle after surgery.Surgical treatment of endometriosis does not alter embryo quality in patients with infertility treated with IVF.

Abstract

To investigate the role of fast blastocoele re-expansion in the selection of viable thawed blastocysts for transfer.Retrospective study.Academic assisted reproductive program.Transfer cycles were divided into two groups according to the presence or absence of fast re-expanded blastocysts. In group I (124 cycles), all transferred blastocysts had fast re-expanding blastocoele. In group II (113 cycles), no fast re-expanded blastocysts were included in the transfer.Blastocyst survival was defined as >50% of cells remaining intact after thaw and re-expansion after culture in vitro for 2-4 hours before transfer. Blastocysts with >or=50% re-expansion were designated as fast re-expanded blastocysts.Percentage of blastomere loss immediately after thaw, degree of blastocoele re-expansion, and clinical outcomes (pregnancy and implantation rates).The rates of survival and fast blastocoele re-expansion of partially intact blastocysts were significantly reduced as compared with fully intact blastocysts. Significantly higher rates of clinical pregnancy (37.1% vs. 16.8%) and implantation (26.7% vs. 11.3%) were obtained when all transferred blastocysts had fast re-expanding blastocoele as compared with those transfers without fast re-expanded blastocysts included.Our results showed that blastomere loss of thawed blastocyst was associated with a reduced ability to re-expand. As a discriminative morphologic marker of superior embryo viability, a fast re-expanded blastocyst would be given priority for transfer to better utilize the cryopreserved blastocysts.

Abstract

Hundreds of thousands of human embryos are cultured yearly at in vitro fertilization (IVF) centers worldwide, yet the vast majority fail to develop in culture or following transfer to the uterus. However, human embryo phenotypes have not been formally defined, and current criteria for embryo transfer largely focus on characteristics of individual embryos. We hypothesized that embryo cohort-specific variables describing sibling embryos as a group may predict developmental competence as measured by IVF cycle outcomes and serve to define human embryo phenotypes.We retrieved data for all 1117 IVF cycles performed in 2005 at Stanford University Medical Center, and further analyzed clinical data from the 665 fresh IVF, non-donor cycles and their associated 4144 embryos. Thirty variables representing patient characteristics, clinical diagnoses, treatment protocol, and embryo parameters were analyzed in an unbiased manner by regression tree models, based on dichotomous pregnancy outcomes defined by positive serum beta-human chorionic gonadotropin (beta-hCG). IVF cycle outcomes were most accurately predicted at approximately 70% by four non-redundant, embryo cohort-specific variables that, remarkably, were more informative than any measures of individual, transferred embryos: Total number of embryos, number of 8-cell embryos, rate (percentage) of cleavage arrest in the cohort and day 3 follicle stimulating hormone (FSH) level. While three of these variables captured the effects of other significant variables, only the rate of cleavage arrest was independent of any known variables.Our findings support defining human embryo phenotypes by non-redundant, prognostic variables that are specific to sibling embryos in a cohort.

Abstract

To report the case of a patient undergoing in vitro fertilization (IVF) in which a non-pronuclear (0PN) oocyte resulted in a normal pregnancy.A 36-year-old woman underwent an IVF-embryo transfer treatment cycle.Four oocytes were retrieved for insemination by IVF. Examination for fertilization revealed two polypronuclearpolygynic and two non-pronuclear oocytes. The non-pronuclear oocytes were observed further for development. One embryo developed from the non-pronuclear cohort and was transferred at the 8-cell stage on day 3. Subsequently, a pregnancy developed, and resulted in the delivery of a healthy term infant.Non-pronuclear oocytes may represent a source of developmentally competent embryos, and further observation of this cohort should be considered, particularly in situations involving a low yield of oocytes at retrieval.

Abstract

CD9 mRNA and protein expression levels in mouse slow frozen-rapid thawed oocytes were compared with those in fresh oocytes by using comparative quantitative real time reverse transcription-PCR and semiquantitative Western blot, respectively. The expression levels of both CD9 mRNA and protein in the frozen oocytes were significantly lower than those found in the fresh oocytes.

Abstract

The purpose of our study is to compare the occurrence of monozygotic twinning (MZT) from blastocyst transfer (BT) in our program between an earlier and more recent time period.Retrospective.Academic IVF practice.All pregnancies conceived between March 2002 and December 2005 (N = 932) in our program were compared to pregnancies conceived before March 2002 (N = 554), which were the subject of a previous study.None.The incidence of MZT with day 3 embryo transfer and BT were compared between the study and control groups.During the study period, the rate of MZT was not significantly different for BT at 2.3% (9/385) compared to day 3 embryo transfer at 1.8% (10/547). This rate of 2.3% for BT was significantly lower than the rate of 5.6% (11/197) reported at our institution for BT before March 2002.Our study suggests that the risk of MZT with BT is significantly lower in the more recent time period and is in the range of what is seen with cleavage stage transfer. It is likely that improvements in culture systems as experience is gained with BT played a role.

Abstract

To evaluate the fertilization and developmental potential of immature oocytes obtained from controlled ovarian hyperstimulated cycles of patients undergoing intracytoplasmic sperm injection (ICSI).Retrospective study.Academic assisted reproductive technology program.Two hundred patients with at least one mature oocyte and one immature oocyte (study 1), and 44 patients with no mature oocytes (study 2) at time of oocyte denudation.Oocyte denudation was performed immediately after retrieval. Oocytes were cultured in vitro for 4-6 hours before ICSI and then categorized into four groups: group I, metaphase II (MII) oocytes at denudation; group II, in vitro matured MII oocytes; group III, metaphase I (MI) oocytes that did not progress to MII; and group 4, germinal-vesicle (GV) oocytes that converted to MI.Fertilization and embryo development were compared among groups in study 1. Pregnancy and implantation rates were evaluated in study 2.Although the fertilization rate in group III was significantly lower than in groups I and II, no significant difference was found between groups I and II. Day 3 embryos in group I had the highest mean number of blastomeres, proportions of good embryos, and blastocyst formation rate when compared with groups II and III. Two clinical pregnancies were achieved from 26 transfer cycles in study 2, resulting in pregnancy and implantation rates of 7.7% and 4% per transfer cycle, respectively.Although our results show that immature oocytes from stimulated cycles can be normally fertilized and used to increase the number of embryos available for transfer, the increase in number of embryos derived from immature oocytes cannot be efficiently translated into pregnancies and live births. The clinical significance of using immature oocytes in stimulated cycles needs further investigation.

Abstract

We compared the effects of two standard oxygen concentrations, physiological (5% O(2), 5% CO(2), and 90% N(2)) and atmospheric (5% CO(2) with the balance as air), on fertilization, embryo development, and pregnancy rate in 106 patients undergoing IVF, excluding donor oocyte cycles and preimplantation genetic diagnosis cycles. The differences in oxygen concentration did not significantly affect fertilization rate, blastocyst formation, or pregnancy rate, but there was a significant difference in mean embryo score between physiological and atmospheric groups on day 3.

Abstract

To study whether fecundity was recovered in mice into which umbilical cord blood cells (UCBCs) were transfused after lethal-dose radiation, followed by transplantation of frozen-thawed ovaries.Prospective basic research study.Academic research laboratory.Female C57BL/6 mice as recipients of UCBCs and ovaries, male B6C3F1 mice for mating, and green fluorescent protein (GFP)-transgenic mice: 18.5-day-old fetuses (-/+) for UCBCs and adult GFP mice (+/+) for ovarian tissues.The UCBCs were transfused into each irradiated mouse, with GFP+ ovaries transplanted 4 weeks later. The chimeric mice were mated 3 weeks after ovarian transplantation and were examined 14 to 16 weeks after the transfusion of UCBCs.Percentage of chimerism, number of GFP+ pups.The percentage of chimerism in these mice tends to increase with the radiation dose. The recovery of fecundity was observed in the chimeric mice that were transplanted with fresh and previously vitrified ovaries after exposure to radiation.Even when the exposure dose of radiation administered as pretreatment is lethal, the fecundity of recipients can be maintained if their ovaries are cryopreserved before they are exposed to radiation.

Abstract

To our knowledge no simple, disposable and accurate test of semen quality currently exists. A novel technique to assess the motile sperm concentration of the human ejaculate has been designed and its results are presented.In a micromachined device fluorescently labeled motile sperm traverse a hydrostatic microfluid line to a target detection cuvette. A microfluorometer assesses the fluorescence signal generated by sperm accumulating there throughout a 50-minute study period. A total of 21 semen specimens from men presenting to our university based reproductive endocrinology and infertility center were tested a total of 67 times. Semen parameters determined by computer assisted semen analysis were compared to the signal reported by the microfluidic device.The fluorescence signal increased throughout the data collection period for all samples. Pearson r values relating the device signal to total and progressive motile concentration were 0.79 and 0.80, respectively (each p <0.001). A signal threshold based on the aggregate data were established, correlating with the WHO standard of the normal total motile sperm concentration. As a screening test, the device was 94% sensitive and 97% specific for identifying samples with less than the WHO standard for the total motile concentration, and 96% sensitive and 90% specific when considering the progressive motile concentration.A novel microfluidic device is presented that enables accurate assessment of the motile sperm concentration in human ejaculate compared to computer assisted semen analysis. Its size and design demonstrate the feasibility of applying laboratory on chip technology to male infertility screening.

Abstract

To investigate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the development of preimplantation mouse embryos.Mouse 2-cell embryos were collected and cultured in P-1 medium supplemented with GM-CSF at different concentrations. Using reverse transcription-polymerase chain reaction, expression Bcl-2 and Bax mRNA in blastocyst were evaluated in the GM-CSF group and control group. Apoptosis detection was performed using the in situ apoptosis detection kit in mouse blastocysts. The statistical significance of the data was analyzed using t-test and chi-square test.The development of blastocyst increased to 89% in the addition of GM-CSF (0.125 ng/mL), compared to controlled group (80%). The number of cells staining for apoptosis was lower in GM-CSF group than that in the control group. Bcl-2 expression was found to be upregulated in blastocysts in the GM-CSF supplemented group compared to the control group.These results suggest that GM-CSF might be an important regulator in embryo development.

Abstract

To present a case involving the transfer of a single pronucleated oocyte resulting in a monozygotic twin pregnancy.A descriptive case report of a single patient.The patient conceived and was found to have a monochorionic diamnionic pregnancy which resulted in the birth of normal identical twin boys at 32 weeks of gestation.The case report addresses an issue that has not received proper attention in the literature. It illustrates that observing a single PN in an oocyte at fertilization check should not be an absolute deterrent to transferring the resulting embryo even in an older patient with a high FSH level. This report also suggests that single observations, especially at the assessment of fertilization, in the IVF laboratory are limited when evaluating embryo potential and normalcy.

Abstract

Although in vitro fertilization (IVF) success rates have improved over the past decade, multiple pregnancies have become a formidable problem. The solution to this problem seems simple by mandating the reduction in numbers of embryos transferred. However, this is typically not accomplished without a compromise in the pregnancy rate. There have been a number of approaches designed to address high order multiple pregnancies from multi factorial analysis of early cleavage stage embryos to the development of extended culture systems, both of which require manipulations in the culture environment. Manipulations in embryo culture environment may not be benign. Several studies have demonstrated that adverse culture conditions have effects on gene expression and imprinting. Studies have also demonstrated that singleton human IVF babies have lower birth weight and higher incidence of congenital anomalies than natural conception babies. All of these factors need to be considered in relation to long term viability of IVF babies and the Barker hypothesis.

Abstract

To examine human embryos at various stages of preimplantation development for simultaneous expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs).mRNAs of specific MMPs and TIMPs were examined in single human embryos, at different stages of preimplantation development, by reverse transcription-polymerase chain reaction (RT-PCR). Single embryo immunohistochemistry was applied to examine the protein expression.University-affiliated IVF-ET program.Couples, attending the university-affiliated IVF-ET program, electing to donate poor prognosis embryos with anomalous morphology.None.The expression of MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in preimplantation embryos.The MMP-2 mRNA was expressed consistently during development from one-cell to blastocyst stage. The TIMP-1 and TIMP-3 mRNAs were detected in embryos at all stages; however, in the later preimplantation developmental stages, an increasing proportion of embryos expressing TIMP-1 and TIMP-3 mRNA were noted. The MMP-1, MMP-9, and TIMP-2 mRNAs were detected in only a minority of human embryos studied. Immunohistochemistry showed MMP-1 and TIMP-1 protein expression in preimplantation embryos.The existence of MMP and TIMP mRNA expression in human preimplantation embryos argues for a role for these metalloproteinases and their inhibitors during the process of implantation in humans.

Abstract

To evaluate the sex ratio in births conceived with blastocyst transfer compared to day 3-ET.A retrospective analysis of IVF patients who became pregnant after blastocyst or cleavage stage transfer at Stanford University Hospital and a literature review were performed.In the day 3-ET group, the male-to-female (M/F) ratio was 157/139 (53%/47%) compared to 97/66 (59.5%/40.5%) in the blastocyst group (P = 0.18). Similar trends have been found in individual studies in the literature but reached statistical significance in only one out of six reports reviewed. The combined data from our study and the literature show a male-to-female ratio of 797/594 (57.3%/42.7%) in blastocyst transfer compared to 977/932 (51.2%/48.8%) in day 3-ET (P = 0.001).Although individual studies may lack power to show an altered sex ratio with blastocyst transfer, the combined data presented in this report do suggest that the M/F ratio is higher with blastocyst transfer compared to cleavage stage transfer.

Abstract

To describe a case of primary infertility associated with oocytes having one pronucleus before fertilization on repeated IVF attempts.Case report.A university-based assisted reproduction unit.A 30-year-old woman with primary infertility and oocytes containing one pronucleus before fertilization.Oocyte donation.Pregnancy.Conceived triplets after transfer of three embryos using donor oocytes.This patient's infertility was likely associated with an oocyte abnormality, as evidenced by the premature formation of one pronucleus before fertilization. In the future, more studies on the appearance of a single pronucleus before fertilization will be needed to determine its overall significance on fertility.

Abstract

To investigate whether ICSI (intracytoplasmic sperm injection) results in decreased blastocyst formation and pregnancy compared to IVF (in vitro fertilization).We performed a retrospective analysis of blastocyst transfer (BT) offered routinely to patients under age 40 with > or = three 8-cell embryos on day 3 and compared IVF to ICSI cycles. Sequential media were used with P1 until day 3, then Blastocyst Medium until day 5/6.There were 131 IVF and 75 ICSI cycles. There was no difference in age, number of oocytes, zygotes, 8-cell embryos, blastocysts on days 5 and 6, or embryos transferred. Progression to blastocyst was similar (78% for IVF and 73% for ICSI) as was the viable pregnancy rate (51.4% for IVF and 55% for ICSI). No cycles failed to form blastocysts.The progression to blastocyst and the likelihood of conceiving a viable pregnancy were unaltered by ICSI. Thus it seems appropriate for programs to offer BT to patients undergoing ICSI using the same inclusion criteria applied to their IVF patients.

Abstract

To evaluate the incidence of monozygotic twinning (MZT) in pregnancies conceived after blastocyst transfer compared to cleavage-stage transfer.Retrospective study.University IVF program.All IVF patients with viable pregnancies conceived during a 4-year period.Blastocyst transfer or day 3 ET.Incidence of MZT assessed by transvaginal ultrasound.There were 11 incidences of MZT in 197 viable pregnancies (5.6%) with blastocyst transfer compared to 7 of 357 viable pregnancies (2%) with day 3 ET. In 10 of 18 pregnancies, MZT was observed in the setting of a higher order multiple gestation (6 of 11 for blastocyst transfer and 4 of 7 for day 3 ET). In the day 3 ET group, assisted hatching or intracytoplasmic sperm injection (ICSI) did not increase MZT (4 of 213, 1.9%) compared to cycles without zona breaching (3 of 144, 2.1%). Similarly, in the blastocyst-transfer group, ICSI did not increase the incidence of MZT (4 of 74, 5.5% for ICSI and 7 of 123, 5.7% for non-ICSI IVF).Compared to day 3 ET, blastocyst transfer appears to significantly increase the incidence of gestations with MZT. This information should be taken into account when counseling patients about the pros and cons of extended culture.

Abstract

To compare cycle outcomes in similar populations of women over 40 who underwent blastocyst transfer compared with women who had day 3 embryo transfer with assisted hatching (ET/AH).Retrospective study. STTING: University hospital-based program.Eighty-six IVF cycles in women ages 40 to 43 years who had more than three eight-cell embryos on day 3. On day 3 of embryo culture, patients chose either to undergo blastocyst transfer or day 3 ET/AH.Pregnancy and cryopreservation rates were recorded.In 48 cycles, blastocyst transfer was performed, and in 38 cycles day 3 ET/AH was performed. There was no statistically significant difference between the blastocyst transfer group and the day 3 ET/AH group with respect to age (41.1 +/- 0.9 years vs. 41.6 +/- 0.8 years), percentage of intracytoplasmic sperm injection cycles (29.2% vs. and 27.6%), number of oocytes (14.9 +/- 5.6 vs. 12.8 +/- 4.0), or number of eight-cell embryos (6.1 +/- 2.2 vs. 5.4 +/- 1.5). Significantly fewers embryos were transferred per cycle with blastocyst transfer (2.6 +/- 1.0) compared with day 3 ET/AH (5.9 +/- 2.0). The viable pregnancy rate was similar in the blastocyst transfer group (29.2%) and in the day 3 ET/AH group (26.3%). Embryos for cryopreservation were available in significantly more cycles in the blastocyst transfer group (52.1%) than in the day 3 ET/AH group (21.1%). Cleavage stage arrest occurred only in one cycle.Blastocyst transfer appears to be as effective as day 3 ET/AH in older patients with good embryos. Higher cryopreservation rate in the blastocyst transfer group may represent an advantage over day 3 ET/AH. Older women may also benefit from the information that extended culture provides them regarding their oocyte quality.

Abstract

Our aim was to determine the impact of freezing, thawing, and subcutaneous transplantation on follicular development in grafted mouse ovaries.The mice were divided into 3 groups: control (group 1), frozen-thawed grafting (group 2), and frozen-thawed grafting with human menopausal gonadotropin injection (group 3). After freezing and thawing, the ovaries were transplanted into the subcutaneous tissue. Two weeks after transplantation, grafted ovaries and blood samples were collected.Ovaries from group 3 contained significantly more follicles (246 +/- 43 follicles) than group 2(P

Abstract

Phospholipase A(2) (PLA(2)) and cyclooxygenase (COX) are two key enzymes in PG synthesis; the latter has two forms, COX-1 and COX-2. mRNA was extracted from single preimplantation embryos and examined for PLA(2), COX-1, and COX-2 gene expression by RT-PCR to investigate whether PLA(2) and COX genes are expressed in human preimplantation conceptuses from zygote to blastocyst stage and to compare COX-1 and COX-2 gene expression within the same stage of embryonic development. Expression of PLA(2), COX-1, and COX-2 was detected in 48, 37, and 45%, respectively, of total embryos examined. COX-1 was expressed in approximately 66% of early human preimplantation embryos from zygote to two-cell stage, whereas COX-2 was expressed in about 58% of later stage embryos from eight-cell to blastocyst stage (P < 0.05). Furthermore, COX-2 mRNA and protein were localized to trophectoderm in blastocyst stage embryos. In conclusion, PLA(2), COX-1, and COX-2 are expressed during early human embryonic development and may contribute to the production of PGs such as PGE(2) in human embryogenesis. COX-1 and COX-2 are differentially expressed, with COX-2 being primarily expressed by trophectoderm in late-stage human preimplantation embryos, which may promote embryonic differentiation and implantation.

Abstract

To assess the accuracy of day 3 morphologic criteria in identifying the best embryos.Prospective observational study.University IVF program.One hundred cycles in women desiring blastocyst transfer who had > or =3 eight-cell embryos on day 3.On day 3, the embryologist chose the two embryos that would have been transferred that day. On day 5, embryos were examined to determine the best and second-best blastocysts.Accuracy of day 3 picks as measured in culture on day 5, outcome of nontransferred picks, and cryopreservation rate.All cycles reached the blastocyst stage and 73% had cryopreservation. The mean number of blastocysts was 4.8 (3.2 on day 5 and 1.6 on day 6). Neither pick was chosen in 39% of cycles; one pick was transferred in 38%; and both picks were transferred in 23%. Of 116 nontransferred picks, 51 were frozen and 65 arrested, with both picks arresting in 9 cycles. The single best blastocyst was chosen from the picks in 39% of cycles.Morphologic criteria for cleavage-stage embryo selection may fall short when the transfer is limited to two embryos. Culture to blastocyst is warranted in this population to avoid high-order multiples and still be able to choose the two embryos with the highest implantation potential.

Abstract

To compare implantation and pregnancy rates (PRs) achieved with blastocyst transfer (BT) and day 3 ET in similar patient populations.Retrospective analysis.Academic infertility center.One hundred consecutive patients <40 years undergoing IVF, each with more than three eight-cell embryos on day 3.Patients used their own eggs for IVF or IVF and intracytoplasmic sperm injection. Embryos were cultured in P1 medium (Irvine Scientific, Santa Ana, CA) until day 3, when they were either transferred or, in the case of embryos for BT, incubated in Blastocyst Medium (Irvine Scientific), followed by transferring on day 5.Implantation and PRs.There were no statistically significant differences in patient age, FSH level, or number of oocytes or zygotes. The BT group had fewer embryos transferred (mean, 2.4) compared with the day 3-ET group (mean, 4.6). The viable PR (cardiac activity at 6-7 weeks was considered indicative of a viable pregnancy) was higher with BT (68%, 34/50) than with day 3 ET (46%, 23/50). The implantation rate was increased with BT (47%, 56 sacs/120 embryos) compared with day 3 ET (20%, 46 sacs/231 embryos).The BT group in our study had higher implantation and PRs compared with the day 3-ET group. Better embryo selection, improved embryo-uterine synchrony, and decreased cervical mucus on day 5 may have accounted for the enhanced outcome. Our data support the use of BT to limit the number of embryos transferred while improving PRs.

Abstract

To examine the effect of the number of blastocysts transferred on pregnancy and multiple gestation rates.Retrospective study.Academic infertility center.Patients < 40 years undergoing IVF, with FSH levels of < 15 mIU/mL and more than three eight-cell embryos.Embryos were cultured in P1 until day 3 and then transferred to blastocyst medium. A maximum of three blastocysts were transferred.Pregnancy, multiple gestation, and implantation rates.All 55 patients developed blastocysts and underwent ET. Twenty-four patients had three embryos transferred and 29 patients had two embryos transferred. Two patients had only one embryo each for transfer. There was no difference in the viable pregnancy rate between the two-blastocyst transfer and three-blastocyst transfer groups (62% vs. 58%). In the two-blastocyst transfer group, 39% of pregnancies were multiple gestations (all twin gestations), compared with 79% of pregnancies in the three-blastocyst transfer group (50% twin gestations, 29% triplet gestations). The implantation rate was 47% in both groups.A commercially available, sequential culture system is highly effective for producing viable blastocysts. Two-blastocyst transfer eliminated the risk of triplets while maintaining the same high success rates seen with three-blastocyst ET.

Abstract

This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.

Abstract

Inhibition of sperm phosphodiesterase (PDE) has been shown to increase cAMP concentrations and stimulate motility and the acrosome reaction. While several PDE genes exist in mammals, little is known about the physiological role of PDE forms expressed in human spermatozoa. Using type-selective inhibitors, we identified two of the PDE forms expressed in human spermatozoa and studied their involvement in sperm function. Selective inhibitors of calcium-calmodulin-regulated PDE1 (8-methoxy-isobutyl-methylxanthine) and cAMP-specific PDE4 (RS-25344, Rolipram) were used to study PDE forms in human sperm extracts. 8-MeIBMX and Rolipram/RS-25344 inhibited sperm PDE activity by 35-40 and 25-30% respectively. Subcellular fractionation of the sperm homogenate suggests these pharmacologically distinct forms may be located in separate cellular regions. To evaluate the functional significance of different PDE forms, the effect of type-specific PDE inhibition on sperm motility and the acrosome reaction was examined. PDE4 inhibitors enhanced sperm motility over controls without affecting the acrosome reaction, while PDE1 inhibitors selectively stimulated the acrosome reaction. These data indicate at least two distinct PDE types exist in human spermatozoa. Our findings also support the hypothesis that PDE subtypes affect sperm function by regulating separate pools of cAMP and may ultimately offer novel treatments to infertile couples with abnormal semen parameters.

Abstract

Gaining knowledge about the physiological timetable of gene expression during preimplantation embryo development is crucial, and a better understanding of cytokine and growth factor expression in early embryonic development could lead to improved in vitro culture conditions and enhance in vitro fertilization implantation rates. Our aim was to detect the patterns and levels of two messenger ribonucleic acids [mRNAs; beta-actin and interleukin-1 receptor type I (IL-1R tI)] in single human blastomeres by RT-nested PCR and to compare possible variations in the gene expression both between different embryos and in multiple blastomeres within the same embryo. Single blastomeres from nine human tripronucleic preimplantation embryos were examined by one round of RT and two rounds of nested competitive PCR. Beta-actin mRNA was detected in each blastomere, and IL-1R tI mRNA was found in 72% of the blastomeres examined. Beta-actin was expressed at a level of 511-12185 molecules of complementary DNA/blastomere, and IL-1R tI was expressed at a level of 2-290 molecules of complementary DNA/blastomere. Our results suggest that the mRNA pattern of an embryo cannot be reliably quantitated from the mRNA pattern of a single blastomere and therefore imply limitations for the use of this method for preimplantation diagnosis.

Abstract

To review the authors' experience in a successful frozen blastocyst program.Retrospective study.University IVF program.Women of all ages undergoing 64 frozen blastocyst nondonor thaw cycles.Thaw cycles with day 5 or day 6 frozen blastocysts replaced into luteal day 5 endometrium in natural or programmed cycles; cryopreservation of blastocysts by Menezo two-step protocol and Testart slow cool program; thawing by two step thaw protocol.Implantation, clinical pregnancy, and delivery rates.The implantation rate was 16% and was similar with day 5 frozen blastocysts and day 6 frozen blastocysts cycles. The clinical pregnancy rate was 36% and the delivery rate was 27%, with no significant difference between day 5 and 6 blastocysts.Blastocyst cryopreservation is a viable option for patients of all ages and complements fresh blastocyst culture and transfer. The presence of good quality blastocysts for freezing on day 5 and day 6 yields comparable results and is critical for the success of thaw cycles.

Abstract

The aim of our study was to detect and characterize mRNA expression of VEGF isoforms VEGF(121), VEGF(145), VEGF(165), VEGF(189), and VEGF(206) in human blastocysts. We recently demonstrated VEGF mRNA expression during human preimplantation embryo development, and further information regarding the alternatively spliced mRNAs resulting in freely secreted proteins or proteins bound to cell surface heparan-sulphate proteoglycans is needed to better understand the process of angiogenesis during implantation. Human blastocysts unsuitable for transfer obtained from the IVF programme at Stanford University were examined by reverse transcription/hemi-nested polymerase chain reaction for their expression of VEGF mRNA splice variants. VEGF mRNA was expressed in 17 out of 19 (89%) blastocysts. Of the 17 blastocysts, VEGF(121) mRNA was detected in 88%, VEGF(145) mRNA in 100%, VEGF(165) mRNA in 71%, and VEGF(189) mRNA in 24% of blastocysts. There was co-expression of mRNA for VEGF(121) and VEGF(145) only in 29% blastocysts, of mRNA for VEGF(165) and VEGF(145) only in 12%, and of mRNA for VEGF(121), VEGF(145) and VEGF(165) in 59% blastocysts. VEGF(206) mRNA could not be detected. In conclusion, we demonstrated that blastocysts express the mRNAs encoding for the free VEGF proteins, enabling the implanting embryo to immediately induce angiogenesis at the implantation site.

Abstract

To detect the expression of vascular endothelial growth factor (VEGF) mRNA and/or secretion of VEGF protein by human preimplantation embryos.Human preimplantation embryos not suitable for uterine transfer were examined for beta-actin and VEGF mRNA expression. Culture media from normally fertilized and developing preimplantation embryos were assessed for VEGF protein secretion.Clinics and academic research laboratories at the Departments of Obstetrics and Gynecology at the Stanford University, Palo Alto, California and the Heinrich-Heine-University, Düsseldorf, Germany.Couples undergoing IVF by intracytoplasmic sperm injection for various reasons.Six unfertilized oocytes and 33 pathologically fertilized (tripronucleic, 3PN) preimplantation embryos were examined for VEGF mRNA expression, and 16 embryos were examined for VEGF protein secretion.Embryonic expression of VEGF mRNA and VEGF protein as determined by reverse transcription (RT)/nested polymerase chain reaction (PCR) and ELISA.VEGF mRNA and protein could not be detected in unfertilized oocytes. However, 30/33 preimplantation embryos did express VEGF mRNA (11/12 10-to-16-cell embryos, 3/4 morulae, 11/12 early blastocysts, 5/5 hatched blastocysts). The VEGF protein level was below the sensitivity of the ELISA.Production of VEGF may give the embryo the ability to induce neoangiogenesis at the implantation site, thus creating an environment necessary for its survival.

Abstract

To examine the rate of monozygotic twinning associated with blastocyst transfer using commercially available, cell-free culture systems with unmanipulated blastocysts.A retrospective analysis was conducted in multiple private and academic infertility centers throughout the United States, of 199 pregnant patients following in vitro fertilization (IVF) blastocyst embryo transfer (ET). Human embryos obtained through standard IVF stimulation protocols were cultured in commercially available, cell-free media systems and transferred as blastocysts. The main outcome measure was the rate of monozygotic twinning.A total of 199 blastocyst-ET pregnancies were achieved during the study period at the fertility centers examined. Monozygotic twinning was noted in 10/199 (5%) of these pregnancies. All were monochorionic diamnionic.Monozygotic twinning previously has been reported following IVF, especially in relation to assisted hatching. While blastocyst transfer has been available for many years using coculture, there have been no published multicenter reports of monozygotic twinning associated with unmanipulated blastocysts. In a multicenter analysis, a definite increase in monozygotic twinning was seen following blastocyst-ET. We believe this phenomenon is real and that this information should be considered when counseling patients for treatment.

Abstract

To examine the IVF day 3-ET pregnancy rate in patients under 40 with sibling embryo blastocyst development, compared with similar patients without blastocyst formation.Retrospective analysis.Academic infertility center.One hundred twenty-five IVF day 3-ET patients under 40 with sibling embryos for extended culture.Extended culture of nontransferred sibling embryos for blastocyst development.Pregnancy and multiple gestation rates, number of oocytes, embryos formed, and embryos transferred.Thirty-eight percent of patients became pregnant. Forty-eight percent of patients had sibling embryos develop to blastocyst. The blastocyst group had more oocytes retrieved (17.4+/-6.6 versus 14.4+/-5.6), more embryos formed (11.2+/-4.2 versus 8.8+/-3.2), and a higher clinical pregnancy rate (60% versus 18%) than the group without blastocyst development.Blastocyst transfer has been shown to improve implantation rates and reduce the risk of multiple gestations from assisted reproductive technology. Sibling embryo blastocyst development may reflect superior embryo quality, as manifested by increased IVF-ET pregnancy rates. In addition to predicting pregnancy in the current cycle, sibling embryo blastocyst development may provide information about the potential for fresh blastocyst transfer in subsequent cycles and help to identify patients at risk for multiple gestations.

Abstract

Hexokinase (HX), the enzyme that catalyses the initial reaction in glycolysis, is an important enzyme in glucose metabolism during human and mouse embryonic development. In our previous investigations of the genetic activities of HX, we observed an increased incidence of HX gene expression in blastocysts in comparison with morulae, and variability in the incidence of HX gene expression between embryos at the same developmental stages. These observations prompted us to quantify HX mRNA in mouse blastocysts to define the biological significance of the variable gene transcription. We modified our qualitative reverse transcription-nested polymerase chain reaction (RT-nPCR) assay for HX mRNA in single or groups of embryos to quantify HX mRNA by competitive RT-nPCR. HX mRNA was quantified in cohorts of mouse blastocysts cultured in glucose/phosphate-containing human tubal fluid (HTF) media. These blastocysts expressed HX in minute amounts, averaging 1.95 x 10(-18) g of mRNA. This is the first attempt at quantification of single gene mRNA in preimplantation embryos. Further investigations using similar techniques will enable comparative analyses between embryos to be performed to determine the correlation between specific levels of HX mRNA transcripts in individual embryos and embryonic viability and competence for further development and implantation.

Abstract

In mouse and human preimplantation development, pyruvate is consumed preferentially during early embryogenesis; however, during the morula and blastocyst stages, glucose is the preferred energy substrate. Studies have suggested that the glycolytic enzymes, hexokinase and glucose phosphate isomerase, are important enzymes in glucose metabolism during these later stages of human and mouse preimplantation development. In order to investigate the genetic activities of these enzymes in late-stage mouse embryos developing in vitro, we analysed hexokinase and glucose phosphate isomerase transcription activities by qualitative RNA assays using reverse transcriptase-nested polymerase chain reaction amplification of individual mouse morulae and early blastocysts incubated in glucose/phosphate-free preimplantation stage one (P1) medium and glucose/phosphate-containing human tubal fluid (HTF) medium. We observed an increased incidence of hexokinase transcripts in the population of blastocysts compared with morulae, and differences in transcript incidence between early blastocysts developing in HTF medium and in P1 medium. In contrast, glucose phosphate isomerase transcripts were consistantly present in all embryos analysed, and appear to be constitutively expressed during late-stage mouse embryogenesis. The different activity patterns of the two glycolytic genes may reflect different mechanisms of gene regulation or differential transcript stability during the later stages of mouse preimplantation development.